Background
Transient global cerebral ischemia is one of the major complications of clinical emergencies such as cardiac arrest, drowning or severe systemic hypotension during a surgical procedure. Currently, the most adequate treatment for these patients is re-establishing perfusion of the brain as soon as possible. However, reperfusion may paradoxically exacerbate brain injury, which is called cerebral ischemia/reperfusion(I/R) injury [
1]. Therefore, efforts need be made that not only preserve cerebral blood flow, but also prevent the actual mechanisms that trigger brain damage after I/R injury [
2].
Neuroinflammation, which is characterized by microglial and astroglial activation, as well as the release of cytotoxic agents (cytokines, matrix metalloproteinases, nitric oxide and reactive oxygen species) can be triggered by cerebral I/R injury, which can contribute to blood–brain barrier disruption and delayed neuronal death [
3]. Subsequently, these damaged cells release more toxic mediators, which in turn activate more immune cells. Thus, prolonged inflammation caused by this vicious circle exacerbates brain damage. Taken together, anti-inflammation therapy may become a promising therapeutic strategy for the treatment of cerebral I/R injury [
3,
4].
The P2X7 receptor(P2X7R), a purinergic receptor, was first discovered in macrophages. In the central nervous system (CNS), the P2X7R is predominantly expressed in microglia which are the resident macrophages of the brain [
5]. The P2X7R can be activated by high concentrations of ATP. Stimulating the P2X7R leads to microglial activation, reactive oxygen species production and increased secretion of pro-inflammatory cytokines such as IL-1ß, TNF-α and IL-6 [
6,
7]. Recently, the P2X7R has been reported to be involved in neuroinflammation in many CNS diseases including Alzheimer’s disease (AD), epilepsy, spinal cord injury and multiple sclerosis, and treatment with P2X7R antagonists reduces experimentally induced neuroinflammation in animal models of such diseases [
6,
8‐
12].
The P2X7R has also been reported to participate in cerebral ischemic injury.
In vitro and
in vivo studies have shown that inhibition of P2X7Rs reduced oxygen and glucose deprivation-induced oligodendrocyte death [
13] as well as infarct volume after transient middle cerebral artery occlusion(MCAO) injury [
14,
15]. However, ischemic injury exacerbation by P2X7R antagonists has also been reported [
16,
17]. To date, the contribution of the P2X7R to cerebral ischemic injury remains an issue, and whether inhibition of P2X7R has beneficial or harmful effects in global cerebral I/R injury has not been studied. We, therefore, designed experiments using two widely used P2X7R antagonists, Brilliant blue G (BBG) and adenosine 5′-triphosphate-2′,3′-dialdehyde (OxATP) [
6], and the selective P2X7R antagonist A-438079 [
18] to investigate the role of P2X7R in a rat model of transient global cerebral I/R injury. We also explored the association between the P2X7R and neuroinflammation after transient global cerebral I/R injury.
Methods
Animals and surgical procedures
Male Sprague–Dawley rats weighing 260–320 g were provided by the Animal Center of Zhejiang University. All procedures used in this study were carried out according to the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and have been approved by the Ethics Committee for the Use of Experimental Animals in Zhejiang University.
Twenty minutes of global cerebral ischemia was induced by the four-vessel occlusion (4-VO) method with slight modification, as established by Pulsinelli [
19], and routinely used in our laboratory [
20,
21]. Briefly, anesthesia was induced with 4% (w/v) choral hydrate (400 mg/kg, intraperitoneally (i.p.)), then the bilateral common carotid arteries (CCAs) were freed and both vertebral arteries were permanently electrocauterized. Rats were allowed to recover for 24 hours after closing the surgical incisions.
On the following day (+0D), anesthesia was applied, the surgical incision in the neck was opened and both CCAs were occluded with aneurysm clips to induce global cerebral ischemia. The clips were removed for reperfusion. Rectal temperature was maintained at 36.5 to 37.5°C throughout the procedures. Cerebral blood flow (CBF) before and after clamping the bilateral CCAs was monitored using a laser Doppler blood flow monitor(PeriFlux System5000, Perimed, Sweden), and rats with a decrease in CBF of less than 80% were excluded [
22].
Drug administration and experimental groups
To study the neuron survival rate in the hippocampal CA1 region after transient global cerebral I/R injury, rats were divided into eight groups: sham group (sham operated), saline group (I/R + saline 2μL intracerebroventricular (i.c.v.), BBG (Sigma, St. Louis, MO) 1 μg, 5 μg, and 10 μg groups (I/R + BBG 1 μg/5 μg/10 μgi.c.v.), OxATP (Sigma, St. Louis, MO) 1 μg, 5 μg, and10 μg groups (I/R + OxATP 1 μg/5 μg/10 μgi.c.v.) and A-438079 (Tocris Bioscience, Ellisville, MO) 0.03 μg, 0.3 μg, and 3 μg groups (I/R + A-438079 0.03 μg/0.3 μg/3 μgi.c.v.). For the other studies, rats were divided into five groups: sham group, saline group, BBG 10 μg group(I/R + BBG 10 μg i.c.v.), OxATP 1 μg group (I/R + OxATP 1 μg i.c.v.) and A-438079 3 μg group (I/R + A-438079 3 μg i.c.v.).
Drug or saline was injected into the right cerebral ventricle (AP −0.92 mm, ML 1.5, DV 3.5 mm) using a microinjector. Drugs were administered into the lateral cerebral ventricle 10 minutes prior to global cerebral ischemia with a total volume of 2 μL at a speed of 0.5μL/minute.
Sample preparation
After three (+3D) or seven days (+7D) of reperfusion, rats were anesthetized and perfused intracardially with saline, followed by 4%(w/v) paraformaldehyde in 0.1 mol/L PBS, pH = 7.4. Brains were removed and fixed overnight in 4%(w/v) paraformaldehyde. Brains were embedded in paraffin, and cut into 4 μm coronal sections at the level of the bregma for H&E staining or immunohistochemistry.
Morris water maze
At +8D, spatial learning and memory were tested using the Morris water maze [
23] which was a circular tank of 120 cm in diameter and 50 cm in height. The tank was filled to a depth of 30 cm with water at 25 ± 1°C. The water was made opaque by adding Chinese ink. The maze was divided into four equal quadrants. The trials were performed according to Vorhees’ method [
24].
Spatial acquisition: All rats received a training trial consisting of daily sessions of four consecutive trials for five days. The hidden platform (diameter 10 cm, 1.5 cm below the water surface) was positioned in the middle of the southwest (SW) quadrant for all rats. The rats were released into the tank facing the maze wall at north (N), west (W), south (S), or east (E) quadrants in a predetermined pseudorandom order. A trial was terminated as soon as the rat found the platform; if the rat did not succeed within 120 seconds, it was guided onto the platform with a stick. The rat was allowed to stay on the platform for 20 seconds before being removed.
Probe trial: Immediately after the final training trial, the platform was removed. Rats were released into the pool at NE position and allowed to swim freely for 2 minutes. The time needed to find the platform (escape latency) in the training trials and time spent in the SW quadrant in the probe trial were recorded. The mean value of four escape latencies in the daily four training trials was taken as the escape latency for the rat. Values from eight rats in the same group were averaged to generate a mean escape latency for that day. Brains of rats in the behavioral study were removed after two weeks of reperfusion and stained with H&E, and the surviving neurons were counted.
Immunohistochemistry
Immunohistochemistry was performed according to the protocol of Wang
et al.[
20]. Briefly, sections were prepared at +3D using the same method mentioned in ‘sample preparation’. Sections were dewaxed with xylene and dehydrated by ethanol at graded concentrations and distilled water. Sections were then incubated for 10 minutes in 3%(v/v) hydrogen peroxide to block endogenous peroxidase activity. High-temperature antigen retrieval was performed in 0.01 M citrate buffer, pH 6.0, for 20 minutes. The brain sections were incubated overnight at 4°C with rabbit anti-Iba1 (ionized calcium-binding adaptor molecule 1), immunoglobulin G(IgG) (diluted 1:500, Wako, Osaka, Japan) or mouse anti-GFAP (glial fibrillary acidic protein) IgG (diluted 1:50, Millipore, Bedford, MA, USA) in PBS containing 0.3%(v/v) Triton X-100, followed by incubation with EnVision (Zymed, San Francisco, CA, USA) solution at 37°C for 30 minutes. Finally, sections were incubated with peroxidase substrate diaminobenzidine until a desired staining intensity developed, followed by slight counterstaining with hematoxylin, dehydration, and cover-slipping with permount. Between incubations, the tissue was washed with PBS three times for 10 minutes each.
In situ labeling of DNA fragmentation (terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)) was preformed with an in situ cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions.
RT-PCR
Hippocampi were rapidly isolated at +3D and total RNA was extracted with TRIzol reagent(Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. RNA (1 μg) was reverse transcribed using random primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA) . The resulting cDNA amplification was performed using the 7500 Fast Real-Time System (Applied Biosystems, Foster City, CA, USA) in conjunction with the SYBR Premix Ex Taq
TM II kit (Takara, Japan). The endogenous housekeeping gene, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), was used as a loading control to normalize target gene expression. Each sample was analyzed in triplicate. Relative expression levels of the target cDNAs were quantified by the 2-ΔΔCts method. The target genes and the specific primers are shown in Table
1.
Table 1
Details of primers (forward & reverse) for different genes used for RT-PCR analysis
IL-1ß | forward | 5′-GCTGTGGCAGCTACCTATGTCTTG-3′ |
| reverse | 5′-AGGTCGTCATCATCCCACGAG-3′ |
TNF-α | forward | 5′-ATACACTGGCCCGAGGCAAC-3′ |
| reverse | 5′-CCACATCTCGGATCATGCTTTC-3′ |
IL-6 | forward | 5′-CCACTTCACAAGTCGGAGGCTTA-3′ |
| reverse | 5′-GTGCATCATCGCTGTTCATACAATC-3′ |
Quantitative analysis
Neuronal damage and apoptosis were quantified by counting the number of surviving neurons or immunopositive neurons for TUNEL in the hippocampal CA1 layer. Photomicrographs of the CA1 region were taken using a digital camera (Olympus DP20, Tokyo, Japan) connected to an inverted microscope (Olympus BX51, Tokyo, Japan). The numbers of surviving/TUNEL-positive neurons and total neurons in the hippocampal CA1 layer per 1 mm length were counted and analyzed by blinded investigators. The severity of neuronal damage/apoptosis was evaluated in the form of neuron survival rate/TUNEL-positive neuron rate which was calculated by the number of surviving or TUNEL-positive neurons/the number of total neurons.
To quantify glial activation, we measured the value of the integrated optical density (IOD) for Immunohistochemistry images from the CA1 region stained with antibodies against Iba-1 or GFAP using Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA). Pictures were taken using the same method as mentioned above. Three high power (400×) images were randomly selected for each animal, and the mean IOD of these three fields was considered as the IOD of the animal.
Statistical analysis
Values are expressed as the mean ± S.E.M. The Kaplan-Meier method was used in the analysis for survival and data were compared using the log-rank test (GraphPad Prism 5). For analysis of escape latency in the behavioral study two-way analysis of variance(ANOVA) was performed. Other data were subjected to one-way ANOVA and post-hoc comparisons were carried out using Bonferroni’s test. Values of P < 0.05 were considered significant.
Discussion
In this study, we demonstrated for the first time that inhibiting P2X7Rs protects against transient global cerebral I/R injury via modulating inflammatory responses in the rat hippocampus. When BBG and OxATP, two of the most widely used P2X7R antagonists, and A-438079, a selective P2X7R antagonist, were centrally administrated right before transient global cerebral I/R injury, they (1) reduced mortality, neuronal cell death and behavioral deficits, and (2) reduced the inflammatory responses as evidenced by a reduction in microglial and astroglial activation, and decreased inflammatory cytokine expression.
Cerebral ischemia rapidly increases inflammatory responses in the rodent brain, which is characterized by astroglial and microglial activation and inflammatory cytokine release [
2,
25‐
27]. Transient global cerebral I/R leads to selective tissue damage in the hippocampal CA1 region, and neuronal death in the CA1 region after global cerebral ischemia has occurred in a delayed manner [
27,
28]. In our present study, apparent neuronal death was observed in the hippocampal CA1 region in the saline group after three to seven days of reperfusion, accompanied by marked glial activation and cytokine overexpression (IL-1ß, TNF-α and IL-6). Astroglial and microglial activation in the hippocampus not only induces the production of inflammatory cytokines but also reactive oxygen species, chemokines, proteases, and vasoactive mediators many of which are cytotoxic to neuronal cells [
29]. Taken together, our findings proved that neuroinflammation following transient global cerebral I/R injury is an important contributor to I/R-induced hippocampal CA1 neuron death.
The P2X7R is predominantly expressed by microglial cells in the CNS. Numerous literature reports have shown that P2X7R stimulation is related to microglial activation, high doses of ATP that elicit microglia proliferation [
30,
31] and morphological transformation [
31], as well as superoxide production [
32] and inflammatory cytokine secretion [
33,
34] which could be inhibited by P2X7R antagonists. Astrocytes normally express low levels of P2X7R [
35‐
37]. However, the expression levels would be elevated in some pathological conditions [
38,
39], thus the astroglial P2X7R may be a direct target of ATP as an immunoregulator. Recently, Jae
et al. [
9] reported that BBG reduced the activation of astrocytes and microglia as well as neuronal death in the hippocampus of amyloid-ß
1–42 (Aß
1–42)-injected rats. Pengetal. [
8] also found that BBG treatment could attenuate glial activation, tissue damage and behavioral deficits in a rat spinal cord injury model. In agreement with these studies, our data showed that BBG, OxATP and A-438079 reduced I/R-induced microglial and astroglial activation, which was accompanied by reduced hippocampal CA1 neuron death, reduced apoptosis, and better spatial memory recovery(Figure
5), These results suggest that P2X7 receptor-mediated microglial and astroglial activation plays a crucial role in neuronal damage in the hippocampal CA1 region following transient global cerebral I/R injury.
Reactive microglia and astrocytes can produce and release various inflammatory cytokines. Our results showed that inhibition of P2X7Rs reduced the expression levels of IL-1ß, TNF-α and IL-6 after transient global cerebral I/R injury. ATP is a potent inducer of IL-1ß release as it activates the P2X7R-caspase 1 pathway [
40]. The P2X7R also mediates TNF-α and IL-6 secretion through diverse signal cascades [
34,
41]. In this study, transient global cerebral I/R injury resulted in a substantial increase in the expression levels of TNF-α, IL-1β, and IL-6 in the rat hippocampus. We noted that in the BBG 10 μg, OxATP 1 μg and A-438079 3 μg treated groups, the levels of pro-inflammatory cytokines were significantly suppressed, which suggests that inhibition of the P2X7R effectively suppresses I/R-induced surges in inflammatory cytokines in the hippocampus.
An increased number of P2X7R antagonists have been identified and optimized by pharmaceutical companies and academic groups. Among them, BBG and OxATP are the most widely used in recent studies of P2X7Rs [
6]. However, both P2X7R antagonists suffer from relatively poor receptor specificity. OxATP has strong antagonistic effects not only on P2X7Rs, but also on other P2XRs [
42,
43], and even P2YRs. BBG has nanomolar affinity on rat P2X7 receptors and only micromolar affinity at some other P2XRs, and used to be the most selective antagonist prior to application of the selective P2X7R antagonist A-438079 in 2007 [
43]. Our results show that all three P2X7R antagonists can protect hippocampal CA1 neurons from global cerebral I/R injury. Rats receiving A-438079 3 μg had higher neuron survival rates and better motor performance than those receiving OxATP 1 μg. This may be due to the complex mechanism of OxATP on other P2Rs, which has not been well studied in cerebral ischemia.
Previous studies of Le Feuvre
et al. [
17] and Yanagisawa
et al. [
16] failed to show any protective effect of OxATP in MCAO models. OxATP is an irreversible antagonist of P2X7Rs, which is widely used in studies of P2X7Rs. OxATP in high concentrations have been reported to be severely cytotoxic to cerebellar granule neurons [
6,
44] and macrophages [
6]. Although an
in vivo study [
10] showed i.c.v. injection of high doses (30 μg/day) of OxATP is safe for rats, the concentration of the injected solution was relatively low (5 mM), and the speed of injection was very slow (1.2 μg/hr). In the study by Yanagisawa
et al. [
16], OxATP was injected (i.c.v) at a concentration of 100 mM (totally 101 μg in 2 μL), which is much higher than the OxATP concentration used in our study (1 mM, totally 1 μg in 2μL). OxATP at high doses and concentrations may be fatal to neuronal cells. Le Feuvre
et al. [
17] used 3 mMOxATP (totally 3 μg in 2 μL) in a mouse MCAO model, and did not observe any protective effect of OxATP. Based on the report of Hibell
et al. [
45], apparent species differences exist in the kinetic properties of P2X7R antagonists, thus the dosage used in Le Feuvre’s study may not be optimal for mice. However, further studies are needed to elucidate the pharmacokinetics of different P2X7R antagonists
in vivo.
Yanagisawa’s study [
16] has shown that 2′, 3′-O-(4-benzoyl-benzoyl)adenosine5′-triphosphate (BzATP), a potent P2X7R agonist protected against MCAO injury in rats. BzATP was widely used in studies of P2X7Rs, as it exhibited at least 10- to 30-fold greater potency than ATP [
46]. However, BzATP was not a selective P2X7R agonist; it could also bind to other P2X receptors, especially P2X1R, P2X2R and P2X3R, and has much more potent effects on P2X1 and P2X3 receptors than P2X7Rs [
6,
47]. Therefore, the mechanisms of BzATP-induced neuroprotection in the rat brain may be far more complicated than activation of P2X7Rs. Unfortunately, to our knowledge, there are no available pharmacological ligands that can selectively activate the P2X7R [
47]. Further research should be aimed at developing P2X7 agonists with greater pharmacologic specificity.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BL, QX and KC conceived the study. BL and QX participated in its design and coordination, KC, GP, JW and MF analyzed the data and drafted the manuscript. KC and BY participated in setting up the model of global cerebral I/R injury. KC, BY and YD performed behavioral testing on the rats. HL and ZX performed the immunohistochemistry and RT-PCR experiments. All authors have read and approved the final manuscript.